Immune Escape Adaptive Mutations in Hemagglutinin Are Responsible for the Antigenic Drift of Eurasian Avian-Like H1N1 Swine Influenza Viruses

Abstract

The continuous antigenic variation of influenza A viruses remains a major hurdle for vaccine selection; however, the molecular determinants and mechanisms of antigenic change remain largely unknown. In this study, two escape mutants were generated by serial passages of the Eurasian avian-like H1N1 swine influenza virus (EA H1N1 SIV) A/swine/Henan/11/2005 (HeN11) in the presence of two neutralizing monoclonal antibodies (mAbs) against the hemagglutinin (HA) protein, which were designated HeN11-2B6-P5 and HeN11-4C7-P8, respectively. The HeN11-2B6-P5 mutant simultaneously harbored the N190D and I230M substitutions in HA, whereas HeN11-4C7-P8 harbored the M269R substitution in HA (H3 numbering). The effects of each of these substitutions on viral antigenicity were determined by measuring the neutralization and hemagglutination inhibition (HI) titers with mAbs and polyclonal sera raised against the representative viruses. The results indicate that residues 190 and 269 are key determinants of viral antigenic variation. In particular, the N190D mutation had the greatest antigenic impact, as determined by the HI assay. Further studies showed that both HeN11-2B6-P5 and HeN11-4C7-P8 maintained the receptor-binding specificity of the parent virus, although the single mutation N190D decreased the binding affinity for the human-type receptor. The replicative ability in vitro of HeN11-2B6-P5 was increased, whereas that of HeN11-4C7-P8 was decreased. These findings extend our understanding of the antigenic evolution of influenza viruses under immune pressure and provide insights into the functional effects of amino acid substitutions near the receptor-binding site and the interplay among receptor binding, viral replication, and antigenic drift. IMPORTANCE The antigenic changes that occur continually in the evolution of influenza A viruses remain a great challenge for the effective control of disease outbreaks. Here, we identified three amino acid substitutions (at positions 190, 230, and 269) in the HA of EA H1N1 SIVs that determine viral antigenicity and result in escape from neutralizing monoclonal antibodies. All three of these substitutions have emerged in nature. Of note, residues 190 and 230 have synergistic effects on receptor binding and antigenicity. Our findings provide a better understanding of the functional effects of amino acid substitutions in HA and their consequences for the antigenic drift of influenza viruses.

Keywords: EA H1N1; HA protein; antigenicity; escape mutant; swine influenza virus.

FIG 1

Homology model of HA and location of the mutated amino acid residues in the HA structure. (A) One promoter of the trimeric HA, with HA1 in cornflower blue, HA2 in purple, and the three mutated residues shown as gold spheres. The other two HAs are in gray. The 190 helix, 130 loop, 150 loop, and 220 loop of the receptor-binding site are labeled and shown in red. (B) The surface of the HA trimer. Highlighted sites are Sa (pink), Sb (cornflower blue), Ca1 (cyan), Ca2 (orange), and Cb (forest green).

FIG 2

Receptor-binding properties of the indicated viruses. The receptor-binding specificity was tested using two different glycopolymers: an α-2,3-sialylglycopolymer (Neu5Acα2-3Galβ1-4GlcNAcβ1-pAP [para-aminophenyl]-α-PGA [α-polyglutamic acid]) and an α-2,6-sialylglycopolymer (Neu5Acα2-6Galβ1-4GlcNAcβ1-pAP-α-PGA). Chicken antisera against the indicated viruses were used as the primary antibodies, and a horseradish peroxidase-conjugated goat anti-chicken antibody was used as the secondary antibody. The absorbance was measured at a wavelength of 490 nm. The data are presented as the means ± SD from three replicates. (A) Wild-type HeN11 virus. (B) Escape mutant virus HeN11-2B6-P5. (C) Escape mutant virus HeN11-4C7-P8. (D) Rescued virus rHeN11/N190D. (E) Rescued virus rHeN11/I230M.

FIG 3

Replication kinetics of wild-type HeN11 and mutant viruses in vitro. MDCK cells and hTERT-PTECs were infected at multiplicities of infection of 0.001 and 0.01. Supernatants were collected at 12, 24, 36, 48, 60, and 72 hpi, and the virus titers were determined by a TCID₅₀ assay in MDCK cells. (A and B) The wild-type HeN11 virus and two escape mutants in MDCK cells and hTERT-PTECs. (C and D) The three rescued viruses in MDCK cells and hTERT-PTECs. Each data point on the curve indicates the mean ± SD from three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. The dashed lines indicate the lower limits of detection.

FIG 4

Polymorphism analysis of the mutated residues in the escape mutants of the HeN11 virus. A total of 768 H1 HA sequences of influenza viruses, including branches of 1C1, 1C1-2-like, 1C2, 1C2-like, 1C2.1, 1C2.2, 1C2.2-3-like, and 1C2.3, isolated from 2001 to 2021 were downloaded from the Influenza Research Database (https://www.fludb.org/brc/home.spg?decorator=influenza [up to 29 November 2021]). These sequences were then subjected to multiple-sequence alignments of the corresponding regions of the HA proteins by using the MAFFT multiple-sequence alignment program (https://mafft.cbrc.jp/alignment/software/). X indicates amino acid residues at the indicated positions other than those found in the HeN11 parent virus and escape mutants.